A biosensor is a device for measuring the concentration of an analyte in a biological sample. A typical biosensor comprises a sensitive biological recognition element able to interact specifically with a target analyte, and a transducer or detector element that is able to transform the recognition event of the analyte with the biological element into a measurable signal. In contrast with conventional bioassays, biosensors allow the detection of molecular interactions as they take place, without requiring auxiliary procedures, making them highly attractive for biotechnological applications.
Among the various types of biosensors, electrochemical biosensors are typically based on enzymatic catalysis of a reaction that produces or consumes electrons. The biosensor substrate usually contains three electrodes: a reference electrode, a working electrode and a counter electrode. The target analyte is typically involved in a reaction that takes place on the working electrode surface, and the reaction may cause either electron transfer across a double layer (producing a current) or can contribute to a double layer potential (producing a voltage).
One such target analyte typically detected using electrochemical biosensors is creatinine. Creatinine is the end metabolite within the human body when creatine becomes creatine phosphate and is used as an energy source for muscle contraction. The creatinine produced is filtered by the kidney glomeruli and then excreted into the urine without reabsorption. The determination of creatinine in body fluids is useful for diagnosing muscle diseases or various kidney diseases such as nephritis and renal insufficiency.
Typically, in order to detect creatinine using an electrochemical biosensor the creatinine needs to be reduced to a detectable product such as hydrogen peroxide. One such pathway for achieving a detectable product includes the enzyme cascade comprising three enzymes (i) creatinine amidohydrolase (CNH) or creatininease, (ii) creatine amidinohydrolase (CRH) or creatinase, and (iii) sarcosine oxidase (SOX). More specifically, the cascade includes using creatinine amidohydrolase to catalyze the hydrolysis of creatinine to creatine. Thereafter, creatine amidinohydrolase may be used to catalyze the hydrolysis of creatine to sarcosine and urea. Finally, sarcosine oxidase may be used to catalyze the oxidative demethylation of sarcosine to yield glycine and detectable hydrogen peroxide. However, as there is a significant concentration of endogenous creatine in the blood, the endogenous creatine significantly effects the determined concentration of creatinine because it is an intermediary byproduct of the enzyme cascade used to detect the creatinine. In other words, the endogenous creatine causes interference with the detection of creatinine in the sample because the endogenous creatine is also reduced to the product hydrogen peroxide via the use of creatine amidinohydrolase and sarcosine oxidase.
Accordingly, to overcome the influence of endogenous creatine on the biosensor, a screening layer containing the enzymes creatine amidinohydrolase, sarcosine oxidase, and catalase may be used to reduce the concentration of the endogenous creatine in the sample. For example, a typical creatinine biosensor may use a double layer deposited over a sensor. One layer may be used to enzymatically convert the creatinine to detectable hydrogen peroxide in an enzyme cascade such as the one described above. The other layer may be used as the screening layer to enzymatically remove the endogenous creatine using an enzyme cascade such as the one described above.
Biosensors comprising the aforementioned screening layer typically have a high design complexity and expensive fabrication techniques. Accordingly, the need exists for improved biosensor designs that addresses endogenous creatine interference and for improved processes for making such biosensors.